Tuberculosis (TB) is a common and often lethal infectious disease caused by the Mycobacterium genus of bacteria, typically Mycobacterium tuberculosis (Mtb). Mtb is air transmissible and easily spreads between individuals through respiratory fluid droplets. Most Mtb infections are asymptomatic, latent TB infections (LTBI), however, some of these infections can eventually progress to active TB infections. TB typically infects the pulmonary system of an afflicted individual; however, the disease can also spread through the body causing extrapulmonary TB (EPTB). Left untreated, active TB typically kills more than half the individuals it affects.
It is estimated that one third of the entire global population is currently infected with Mtb. In 2007 alone, there were an estimated 8 million new cases of TB. During the same year, there were an estimated 1.7 million deaths attributed to TB. Active TB is particularly common in low to middle-income countries, accounting for roughly 80% of reported disease cases. Furthermore, the high prevalence of HIV in areas such as sub-Saharan Africa greatly adds to TB's lethality.
Given TB's high prevalence and associated deaths, fast diagnosis and treatment of active TB is of paramount importance. Currently, the two most common methods of detecting Mtb infection are the sputum acid-fast bacilli smear microscopy test (AFB) and the tuberculin skin test (TST).
In AFB smears, sputum is collected from patients and the sample is examined microscopically after a bacterial staining procedure. Although AFB can produce presumptive results in a few hours, it suffers from poor sensitivity. AFB also fails to identify TB patients having little to no Mtb in their sputum or those patients who are unable to produce sputum. This is especially common in young children or HIV infected patients. Further, Mtb replicates slowly, making positive identification of Mtb in cultures lengthy, ranging from days to weeks.
TST is a composite measure of cell-mediated immunity in response to TB antigen (PPD) stimulation, which is injected under the skin of a patient. However, it may take 2 to 3 days before the results can be obtained and frequently delivers false positive or false negative results. Also, this test does not distinguish latent infection from active disease, which is important in a diagnostic setting.
In addition, nucleic acid amplification tests and interferon-gamma based tests have been developed. Although these tests can provide for rapid detection of Mtb, they also suffer from lack of sensitivity and specificity in certain situations. Moreover, both the cost of such tests, as well as the required expertise to perform such tests can be prohibitive.
A critical need exists for novel diagnostic and prognostic assays to identify those at risk for TB and capable of transmitting Mtb to other individuals. The detection of Mtb biomarkers in a patient's bodily fluid (blood, urine, etc.) provides such an opportunity. Exosomes contained within these bodily fluids can serve as an excellent source of Mtb biomarkers.
Therefore, the identification of specific and easily measured exosomal Mtb biomarkers will have a significant impact on global TB diagnosis and treatment. However, due to the complexity of TB disease, identifying a single Mtb biomarker with the required sensitivity and specificity has been a challenge. Here, the applicants disclose a method of identifying new exosomal Mtb biomarkers indicative of disease and using these exosomal Mtb biomarkers in a robust, simple, accurate and cost effective bioassay to diagnose individuals with active TB disease.